Poly(ADP-ribose) immunostaining to detect apoptosis induced by a neurotoxic fragment of prion protein.

Abstract

PrP106-126 is a synthetic peptide representing codons 106-126 of the prion protein, which spontaneously forms amyloid fibrils and exerts neurotoxic effects on primary mouse brain cell cultures. Neurotoxicity by this peptide is commonly used as a model for the neurotoxicity observed in prion diseases and involves the formation of reactive oxygen species which, in turn, can cause DNA damage, including DNA strand breaks. Strand breaks in nuclear DNA can activate poly(ADP-ribose) polymerase to covalently modify nuclear proteins with poly(ADP-ribose). We, therefore, examined by immunofluorescence whether or not PrP106-126 triggers poly(ADP-ribose) formation. We observed strong poly(ADP-ribose) immunofluorescence signals in a fraction of cells, typically arranged in a clustered pattern, by 30-48 h after peptide addition. A few positive cells were also present in untreated cultures. Cell morphology was suggestive of apoptosis, and this was confirmed by positivity in the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay. On the other hand, our immunofluorescence assay did not detect any 'early' activation of poly(ADP-ribose) polymerase in morphologically normal cells that could have resulted from peptide-induced formation of reactive oxygen species. We conclude that poly(ADP-ribose) immunostaining is a convenient and reliable method for visualizing cells undergoing apoptosis induced by PrP106-126.